Antisera against a PEB1 antigen from Campylobacter jejuni

ABSTRACT

An antigenic composition includes antigens obtainable from Campylobacter jejuni and may be used as a vaccine to induce protective antibodies against both Campylobacter coli and Campylobacter jejuni. The antigenic composition, and antisera specific to the antigens can be used to detect Campylobacter coli or Campylobacter jejuni infection. Diagnostic detection kits include the novel antigenic composition or antisera thereto.

This is a continuation of application Ser. No. 07/986,928 filed on Dec.8, 1992, now abandoned, which is a division of application Ser. No.07/612,330, filed Nov. 13, 1990, now U.S. Pat. No. 5,200,344.

BACKGROUND OF THE INVENTION

This invention relates to novel antigenic compositions useful indiagnostic testing for the presence of Campylobacter jejuni ("C.jejuni") or Campylobacter coli ("C. coli") infection, and useful asvaccines for providing immunological protection against such infection.In certain embodiments, antibodies raised against the novel antigeniccompositions may also be used for diagnostic testing for C. coli or C.jejuni.

C. jejuni and C. coli are believed to cause both inflammatory andnon-inflammatory gastroenteritis (Blaser et al., "Campylobacterenteritis," N. Eng. J. Med., 1981; 305:1444-1452). They are recognizedas leading causes of inflammatory diarrhea in the U.S. and otherdeveloped countries (Blaser et al., Campylobacter enteritis in theUnited States: A Multicenter Study," Ann. Intern. Med.,1983;98:360-365), and are also important causes of endemic diarrhealdisease in the developing world (Glass et al., "Epidemiological andClinical Features of Endemic Campylobacter jejuni infection inBangladesh," J. Infect. Dis., 1983;148:292-296). They have further beenrecognized as common causes of traveller's diarrhea. Additionally, inthe U.S. and other developed countries it has been recognized that C.jejuni and C. coli are commonly found in many different types of foodanimals, including cattle, sheep, goats, swine, chickens, ducks andturkeys. Raw milk may be contaminated with C. jejuni. Althoughcontaminating organisms are readily killed by pasteurization, manypersons consume unpasteurized milk, especially in rural areas. Surfacewater may be contaminated with C. jejuni or C. coli, and persons whoconsume such water may become ill. Commercially raised poultry areparticularly susceptible to contamination with these pathogens (Blaseret al., Epidemiology of Campylobacter jejuni Infections, EpidemiologicReviews, 1983;5:157-176). Many of the animals used as pets by humans,including dogs, cats, and birds may be infected with these organisms,may become ill as a result of infection, or may transmit the organismsto humans. For all of these reasons, it is important that accurate andrapid diagnostic tests be developed for detection of the infections.

Because C. jejuni and C. coli, are fastidious to culture, sophisticatedand time-consuming techniques are required to isolate and identify theseorganisms in a microbiology laboratory. Even with optimal technique, thepresent culture systems may still yield falsely negative cultureresults. Improved methods for the rapid and accurate detection of C.jejuni and C. coli infection are needed.

It is known that persons infected with C. jejuni or C. coli developantibodies specific to the organisms (Blaser et al., "Human SerumAntibody Response to Campylobacter jejuni as Measured in anEnzyme-Linked Immunosorbent Assay", Infect Immun., 1984;44:292-298).Numerous studies have determined that prior C. jejuni or C. coliinfection can be recognized in serological assays. Immunological testingcan be inaccurate, however, unless the antigens used include a highconcentration of conserved antigens with both a high affinity and a highspecificity for the antibodies being detected. The presence of antigenswhich are not sufficiently unique to attract only C. jejuni or C.coli-specific antibodies can lead to the formation of non-specificantigen/antibody complexes and therefore to false positive test results.Conversely, antigens which are not common to most C. jejuni and C. colistrains, or which do not produce strong immunogenic responses to most C.jejuni or C. coli-specific antibodies may not bind the C. jejuni or C.coli-specific antibodies of patients infected with certain strains, thusleading to false negative test results. In such cases, the failure ofantigen/antibody complexes to form does not necessarily indicate lack ofinfection but rather an insensitivity of the test system. Adequatesensitivity often coincides with inadequate specificity, and vice-versa.

Studies performed with human volunteers have shown that the priorexposure to experimental C. jejuni infection offers some protectionagainst subsequent C. jejuni disease. (Black et al., "ExperimentalCampylobacter jejuni Infection in Humans," J. Infect. Dis.,1988;157:472-479; Perlman et al., "Humoral Immune Response toCampylobacter jejuni in Human Volunteers," Abstract presented at the87th Annual Meeting of the American Society for Microbiology, Atlanta,Ga., March 1987; Perlman et al., "Immunity to Campylobacter jejuniFollowing Oral Challenge to Volunteers," Abstract presented at theFourth International Workshop on Campylobacter Infections, Goreborg,Sweden, June 1987.) Similarly, individuals with prior exposure tounpasteurized milk and high pre-existing levels of anti-C. jejuniantibodies have appeared to be at decreased risk of illness when exposedto milk contaminated with C. jejuni (Blaser et al., "The Influence ofImmunity on Raw-Milk Associated Campylobacter Infections,"JAMA1987;257:43-46.) However, while this work indicates that exposure towhole bacteria may induce a protective host response, the pathogenicityof live C. jejuni and C. coli limits the utility of a whole cellvaccine. Conversely a vaccine composed solely of purified C. jejuniantigens in an appropriate vehicle, or a genetically engineeredrecombinant vaccine where the C. jejuni antigen was presented by anavirulent bacteria or virus, would require use of an antigen capable ofinducing the desired immune response in most recipients.

Certain surface proteins of C. fetus are disclosed by "Purification andCharacterization of a Family of High Molecular Weight Cell SurfaceProteins from Campylobacter fetus," Biol Chem., 1988;263:6416-6420.

In Miotti, "Rapid Methods for the Molecular Diagnosis of InfectiousDiseases: Current Trends and Applications," Eur. J Epidemiol., 1987;3:356-364, immunological methods are disclosed for detection ofinfectious agents, but Miotti does not teach a satisfactory diagnostictest for C. jejuni or C. coli.

In co-pending U.S. patent application Ser. No. 07/158,003, filed Feb.18, 1988, Blaser et al. disclose antigenic compositions for use indetecting antibodies specific for Campylobacter [Helicobacter] pylori.

Outer membrane proteins of C. jejuni having molecular weights of 29 kDa,30 kDa and 31 kDa are discussed In Blaser et al., "Campylobacter jejuniouter membrane proteins are antigenic for humans," Infection andImmunity, vol. 43, No. 3, pp. 986-93 (March 1984). Several minorproteins (29-31 kDa) were found to be immunogenic by immunoblotting inDunn et al., "Two-Dimensional Gel Electrophoresis and Immunoblotting ofCampylobacter Outer Membrane Proteins," Infection and Immunity, vol. 55,No. 7 pp. 1564-72 (July 1987),

U.S. Pat. No. 4,404,194 discloses that a 90 kDa protein from C. jejunihas immuno-suppressive activity.

U.S. Pat. No. 4,785,086 discloses a DNA probe for detecting C. jejuni.

U.S. Pat. No. 4,882,271 discloses a 300-700 kDa antigen fromCampylobacter pylori and its use in various assays.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide highly specific andhighly sensitive diagnostic tests for the presence of C. jejuni or C.coli infections.

It is another object of the invention to provide antigenic compositionswhich specifically, and with high sensitivity, attract and bind toantibodies specific to C. jejuni or C. coli.

It is another object of the invention to provide a procedure to aid inthe diagnosis of gastrointestinal symptoms which is relativelynon-invasive and causes little patient discomfort.

It is another object of the invention to provide cost-effective clinicaldiagnostic tests for the presence of C. jejuni or C. coli which aresimple to administer in a clinical or home setting, and which may bequickly evaluated, and to provide kits for performing such diagnostictests.

It is another object of the invention to provide a protein vaccine whichinduces high levels of specific antibodies directed against C. jejuniand which protects against natural C. jejuni infection in humans,livestock, poultry, and other animals.

It is another object of the invention to provide a protein vaccine whichinduces high levels of specific antibodies directed against C. coli andwhich protects against natural C. coli infection in humans, livestock,poultry, and other animals.

It is another object of the invention to provide monoclonal orpolyclonal antibodies specific for C. coli and/or C. jejuni, and methodsfor their use in detection of C. coli and C. jejuni.

These and other objects are accomplished by providing the antigeniccompositions, vaccines, antisera, methods and kits disclosed herein. Inone embodiment of the invention, an antigenic composition comprises atleast one of two Campylobacter jejuni and Campylobacter coli-specificantigens, both of said antigens being obtainable by acid extraction ofsurface antigens C. jejuni, one of said antigens (hereinafter "PEB1"which term includes antigen fragments of the natural protein whetherderived from the natural protein or synthetically or recombinantlyproduced) having an apparent molecular weight of 28 kDa (as measured onsodium dodecyl sulfate polyacrylamide gel under reducing conditions) anapparent molecular weight of 28.9±1.0 kDa (as measured by gel filtrationchromatography under native conditions) and an isoelectric point ofabout 8.5, the other of said surface antigens (hereinafter "PEB3" whichterm includes antigenic fragments of the natural protein whether derivedfrom the natural protein or synthetically or recombinantly produced)having an apparent molecular weight of 30 kDa (as measured on sodiumdodecyl sulfate polyacryl amide gel under reducing conditions), and anisoelectric point greater than 9.3, at least one of said acidextractable antigens being present in said antigenic composition at aconcentration higher than that resulting from acid extraction of surfaceantigens from whole cell Campylobacter jejuni. These antigens (PEB1 andPEB3) are highly conserved, and have strong affinity for antibodiesinduced by most animals' immune response to both C. coli and C. jejuniinfection. The antigens display good specificity, showing low affinityfor antibodies other than those specific for C. jejuni or C. coli.Hence, the antigenic compositions tend to complex with antibodiespresent in the systems of most C. jejuni-or C. coli-infected individualsregardless of the specific strain with which such individuals may beinfected. Moreover, these antigens are seldom recognized by antibodiespresent in the body fluids of non-infected individuals.

In preferred embodiments, each acid extractable surface antigen (PEB1and PEB3) is present in the antigenic composition at a concentration,relative to other acid extractable materials of C. jejuni, greater thantwice the natural concentration of the antigen resulting from acidextraction, and preferably greater than four times the naturalconcentration.

Antigenic proteins having substantial homology to said PEB1 and/or PEB3antigens or their fragments may also be used in accordance with theinvention.

PEB1 and/or PEB3 antigens described above may be capable of inducingprotective immunity against both C. jejuni and C. coli when administeredto an animal in a non-virulent manner. Hence, the antigens may be used,in combination with a suitable adjuvant, as a vaccine against future C.coli or C. jejuni infection, both for human and veterinary applications.The vaccination of poultry, for example, may provide the additionalbenefit of avoiding infection in animals which are consumed by humans,thus avoiding one source of human infection.

Unless expressly stated to the contrary, molecular weights reportedherein were calculated from calibration curves based on relativeelectrophoretic migration of the following molecular weight standards(Bio-Rad) on SDS-PAGE under reducing conditions: lysozyme 14,400daltons; soybean trypsin inhibitor 1,500 daltons; carbonic anhydrase31,000 daltons; ovalbumin 45,000 daltons; bovine serum albumin 66,200daltons; phosphorylase B 97,000 daltons; beta-galactosidase 116,250daltons; and myosin 200,000 daltons. Sodium dodecyl sulfatepolyacrylamide gel (hereinafter "SDS-PAGE") was used in a modifiedLaemmli gel system as described by Ames in a mini-slab apparatus(Bio-Rad Laboratories, Richmond, Calif.). (Ames GFL, "Resolution ofBacterial Protein by Polyacrylamide Gel Electrophoresis on Slabs," J.Biol. Chem., 1974;249:634-644). 1-2 microgram samples for wholebacterial cells or 50 ng for purified proteins were applied to the gelafter boiling for 5 minutes in a buffer containing sodium dodecylsulfate (hereinafter "SDS"), dithiothreitol and glycerol. The separatinggel was 12 percent acrylamide and electrophoresis was performed at 200volts for about 40 min. at room temperature. Proteins were resolvedusing the modified silver stain of Oakley et al. (Oakley et al. Asimplified ultrasensitive silver strain for detecting proteins inpolyacrylamide gels. Anal. Biochem., 1980;105:361-363).

In one aspect of the invention, antigenic compositions containing thePEB1 and/or PEB3 antigens described above are used in methods for thedetection of C. jejuni- or C. coli-specific antibodies. In accordancewith these methods, the antigenic compositions of the invention arecontacted with samples such as body fluids suspected of containing C.coli- or C. jejuni-specific antibodies. Following such contacting, knownmethods are used to determine the extent of formation of anantigen/antibody complex comprised of immunoglobulin bound to antigensfrom the antigenic composition of the invention. When formation of thecomplex exceeds a predetermined positive threshold value, the test ispositive for presence of C. jejuni or C. coli-specific antibody.

Preferred techniques for detecting formation of antigen/antibodycomplexes include, but are not limited to, enzyme-linked immunosorbentassay (ELISA), indirect fluorescence assay, latex agglutination, andliposome-based assay. Alternatively, a Western blot technique may beused, in which case the bands are detected by visual inspection, andsubstantial appearance of dark bands may be taken as a positiveindication.

In one preferred embodiment, for example, the antigenic composition ofthe invention is immobilized and contacted with the sample to be tested.After washing away the sample and any antibodies therein which did notbind to the immobilized antigenic composition, standard methods are usedto determine the extent to which any immunoglobulin remains bound to theimmobilized antigens.

The extent of detection of the antigen/antibody complex which should beconsidered a positive signal (i.e., an indication that the test sampleincludes C. jejuni- or C. coli-specific antibody) depends upon thedetection means chosen, but may be defined generically as a valuegreater than the mean plus 1 interval of standard deviation from theresults observed from a negative control group, all other parameters(dilution of sample, time of incubation, etc.) being held constant. Insome embodiments where higher specificity is desired mean plus two ormean plus three standard deviations may be utilized. The negativecontrol group should consist of asymptomatic individuals who are membersof a population which is unlikely to include individuals infected withC. jejuni or C. coli. A preferred control group, for example, is a groupof asymptomatic U.S. children below 10 years of age. Such children forma population unlikely to be infected.

In one aspect of the invention, kits are provided which include bothantigenic compositions within the scope of the invention, and whichfurther include means for detecting the presence of any immunoglobulinin a test sample which may become bound to antigens in said composition.

Antisera raised against the PEB1 and/or PEB3 antigen described above maybe used in a particularly sensitive and specific test for presence of C.jejuni and C. coli. Test samples are contacted with such antisera,followed by detection of antibody binding to components of the testsample. Where such binding exceeds a predetermined positive thresholdlevel, the sample is positive for C. jejuni or C. coli. The threshold isdetermined as described above. Kits containing such antisera and meansfor detecting such antibody binding provide a convenient means ofpracticing the test.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of protein elution over time (and of decreasing sodiumsulfate concentration) from hydrophobic interation FPLC on aphenyl-Superose column (Pharmacia), used for purifying PEB1 antigen fromC. jejuni strain 81-176. Conditions for purification are as described inthe text. Fractions of eluted materials were collected every 2 minutesand were checked for the presence of PEB1 antigen by SDS-PAGE. Peak 2contained a mixture of PEB3 and PEB4 antigens, and Peak 3 contained PEB1antigen with greater than 98% purity.

FIG. 2 is a photograph of protein bands following electrophoresis onSDS-PAGE of purified C. jejuni proteins from strain 81-176. Lanes arewhole bacterial cells(WC), acid extract(AE), PEB1(28K), PEB2(29K),PEB3(30K) and PEB4(31K) antigens. Molecular weight markers are shown atleft.

FIG. 3 is a graph showing recognition of Campylobacter and Helicobacterby antisera to C. jejuni proteins, by ELISA. Whole bacterial cells wereused as antigens. First antibodies were rabbit anti-acid extract. O.D₄₁₄value greater than 0.1 was defined as positive.

FIG. 4 is a graph showing recognition of Campylobacter and Helicobacterby antisera to C. jejuni proteins, by ELISA. Whole bacterial cells wereused as antigens. First antibodies were rabbit anti-PEB1 antigen. O.D₄₁₄value greater than 0.1 was defined as positive.

FIG. 5 is a Western blot of anti-PEB1 against representativeCampylobacter and Helicobacter strains. The antigens used are wholecells prepared as described in EXAMPLES 2 and 3. Bacterial strains of C.jejuni (except strains 81-176, 81-93 and 81-94), C. coli and C. laridishad been identified by DNA hybridization. The C. fetus strains wereidentified by the presence of high molecular weight surface arrayproteins detected by SGS-PAGE and Western blot (Z. Pei and M. Blaser J.Clin. Invest. 86:1036-1043, 1990). The method for the Western blot is asdescribed in the text. The arrow indicates 28 kDa bands, which werefound in all C. jejuni (strains 81-176, 81-93, 81-95, D996 and D1916,lanes a through e) and all C. coli (strains D743, D1035, D130, D126 andD115, lanes f through j) strains, but not found in any of C. laridis(strains D459 and D1014, lanes k and l), C. fetus (strains 84-32 and80-109, lanes m and n) strains, or in H. pylori strain (strain 16-IIA,lane o).

FIG. 6 is a Western blot of anti-PEB1 against proteinase K-digestedwhole cells and glycine extract from C. jejuni and C. coli. Wholebacterial cells of two C. jejuni (strains 86-64 and 86-223, lanes e, f,g and h) and glycine extract of C. jejuni (strain 81-176, lanes i and j)was used in this study. Lanes a, c, e, g and i containes samples thatwere incubated with H₂ O (control); lanes b, d, f, h and j containedsamples that were incubated with proteinase K. For the samples incubatedwith proteinase K the 28 kDa band, as shown by the arrow, disappeared.

FIG. 7 is a graph of native molecular weight versus elution volume on aSuperose 12 column (Pharmacia LKB, Piscataway, N.J.) on a PEB1 antigenfrom C. jejuni and C. coli. Gel filtration chromatography results shownin FIG. 7 confirm that the native molecular weight of PEB1 issubstantially the same as apparent molecular weight derived fromSDS-PAGE analysis. Semilog regression of molecular weights (MW) of fivestandard proteins ranging from 12.4 to 443 kDa (see text) are usedversus their elution volumes (EV), a formula of log₁₀[MW]=5.53305-0.28746[EV] was generated with a correlation coefficient,r=0.99901. FIG. 7 shows part of the regression curve including MWstandards of 150, 29, and 12.4 kDa. PEB1 antigen eluted immediatelyafter carbonic anhydrase (29 kDa) with a calculated MW of 28.9±1.0 kDa.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

A C. jejuni strain, denoted by the inventors' I.D. No. 81-176 wasoriginally isolated from a human child during an outbreak of C. jejunidiarrheal disease and has been shown to be virulent in studies withhuman volunteers and non-human primates. The strain has been maintainedfrozen at -70° C. in brucella broth (BBL Microbiology Systems,Cockeysville, Md.) containing 15% glycerol. It was deposited with theAmerican Type Culture Collection (ATCC) 12301 Parklawn Drive, Rockville,Md. 20852 U.S.A. on Apr. 3, 1990 and has been accorded ATCC Designation55026. The strain is referred to herein as the "Deposited Strain".Permanence of the deposit and ready accessibility thereto is provided inaccordance with U.S. patent law, the Budapest Treaty, and otherapplicable laws and regulations. The deposit will be accessible to thepublic on and after the date of issuance of any U.S. patent arising fromthe present patent application which refers to the deposit.

All restrictions upon availablility of the deposited strain will beirrevocably removed upon granting of any U.S. patent arising from thepresent application.

The PEB1 and PEB3 antigens from the deposited strain are found in all C.jejuni strains we have tested--not just the deposited strain--and eachis useful in the antigenic composition of the invention regardless ofthe source from which it is derived, including derivation from C. coli.

The Deposited Strain was grown on trypticase soy agar with 5% sheepblood (PASCO, Wheat Ridge, Colo.) in a microaerobic atmosphere (5%oxygen, 10% carbon dioxide and 15% nitrogen) at 37° C. for 24 h forthree generations. Protein concentrations were measured using theMarkwell et al. modification of the Lowry method for crude surfaceprotein (Markwell et al. A modification of the Lowry procedure tosimplify protein determinations in membrane and lipoprotein samples.Anal. Biochem., 1978;87:206-210.), and using Quantigold (DiversifiedBiotech, Newton Center, Mass.) for the purified proteins.

Cells were harvested and washed twice with distilled water bycentrifugation at 3500×g for 15 min. To prepare a crude mixture ofsurface structures, the bacterial cells were suspended in 0.2Mglycinehydrochloride buffer, pH 2.2, at a concentration of 0.1 g (wetweight) of cells to 2.5 ml of buffer. Suspensions were stirred at 25° C.for 15 min. and then centrifuged at 11,000×g for 15 min., thesupernatant was retained, and the pH was neutralized with sodiumhydroxide. The preparation was lyophilized and reconstituted withdistilled water. The salt in this preparation was removed using aSephadex G-15 column (Pharmacia, Piscataway, N.J.) with distilled wateras running buffer. The crude mixture was centrifuged at 1,000×g for 15minutes to pellet any insoluble material. For final purification ofPEB1, the supernatant was separated by hydrophobic interactionchromatography performed on a phenyl-superose column (Pharmacia LKBBiotechnology Inc. Piscataway, N.J.) using an FPLC system (Pharmacia).The initial buffer was pH 8.9, 20 mM boric acid, 1.5M Na₂ SO₄ at a flowrate of 0.25 ml/min. A linear gradient with pH 8.9, 20 mM boric acid wasperformed to 0% of Na₂ SO₄ over 60 minutes and the column eluatemonitored for UV absorbance at 280 nm to define protein peaks (FIG. 1).The fractions of protein eluted were checked for the presence of the 28kilodalton (kDa) protein band using SDS-PAGE analysis (FIG. 2). It wasfound that the 28 kDa protein (PEB1) eluted between about 44 and 50minutes at a sodium sulfate concentration of about 390 mM (FIG. 1).

For purification of PEB3 antigen, the acid extracted material wasseparated by cationic exchange FPLC chromatography on a Mono S column(Pharmacia). The running buffer was 50 mM HEPES, pH 9.3 at a flow rateof 1.0 ml/min and the concentration of NaCl was increased linearly from0 to 400 mM over 20 minutes. Each fraction was checked by SDS-PAGE forthe presence of PEB3 migrating at 30 kDa. The PEB3 antigen eluted fromthe column at about 200 mM NaCl. The fraction containing partiallypurified PEB3 was purified to homogeneity on the phenyl-Superose columnusing the same conditions as described above for the purification of thePEB1 antigen. PEB3 eluted from this column at around 450 mM Na₂ SO₄.Purified PEB3 (30 kDa) is shown in FIG. 2.

Determination of the isoelectric point (pI) of the protein antigen wasmade by isoelectric focusing in RESOLVE™ (thin layer agarose gels) withpH ranging between 3 and 10 using the RESOLVE-HB kit (Isolab Inc.,Akron, Ohio) and the silver stain of Willoughby and Lambert (Willoughbyet al. "A Sensitive Silver Stain for Proteins in Agarose Gels," AnalBiochem., 1983;130:353-358). An experient formula of pI versus migrationdistance was generated by using 3 standard proteins of known pI between6.8 and 9.3. These standard proteins were trypsinogen [pI=9.3], L-lacticdehydrogenase [pI=8.3, 8.4, and 8.6 (3 subunits)], and myoglobin [pI=6.8and 7.2 (2 subunits)]. For the PEB1 antigen, the pI was found to beabout 8.5, and for the PEB3 antigen, the pI was greater than 9.3.

The purified proteins were prepared for amino acid analysis andsequencing by dialysis against water and lyophilization. Amino acidanalysis was performed using the method of Jones (Jones B. N., "AminoAcid Analysis and Enzymatic Sequence Determination of Peptides by anImproved o-phthaldialdehyde Precolumn Labeling Procedure," J. Lig.Chromatogr., 1981;4:565-568). It was found that each of these proteinscontained a large percentage of basic amino acids as shown below inTable 1:

                  TABLE 1                                                         ______________________________________                                        AMINO ACID COMPOSITION OF                                                     PEB1 AND PEB3 ANTIGENS OF C. jejuni                                                            Mole %                                                       Amino Acid         PEB 1   PEB 3                                              ______________________________________                                        Polar                                                                         Lysine             30.2    22.9                                               Histadine          0.6     1.4                                                Arginine           2.0     4.0                                                Asparagine/Aspartate                                                                             11.1    11.9                                               Glutamine/Glutamate                                                                              6.6     7.5                                                Serine             3.8     4.6                                                Threonine          4.4     6.7                                                Nonpolar                                                                      Valine             5.5     5.4                                                Methionine         0.4     1.5                                                Isoleucine         5.2     6.0                                                Leucine            7.4     5.1                                                Alanine            9.4     9.3                                                Phenylalanine      3.3     4.6                                                Tryptophan         not determined                                             Proline            not determined                                             Glycine            6.7     6.3                                                Cysteine           0       0                                                  Tyrosine           3.5     2.8                                                ______________________________________                                    

Amino acid sequencing was performed on an Applied Biosystems 470 AProtein Sequencer equipped with the 120A Autoanalyzer using the 03RPTHprogram. 30 of the first 31 amino acid residues of the amino terminus ofPEB 1 and the 34 amino-terminus acids of PEB 3 were defined as shown inTable 2:

                                      TABLE 2                                     __________________________________________________________________________    AMINO TERMINAL SEQUENCE OF                                                    PEB1 AND PEB3 FROM C. JEJUNI 81-176                                           __________________________________________________________________________           1           5              10             15                           PEB1 SEQ.                                                                            Gly                                                                              Glu                                                                              Gly                                                                              Lys                                                                              Leu                                                                              Glu                                                                              Ser                                                                              Ile                                                                              Lys                                                                              Ser                                                                              Lys                                                                              Gly                                                                              Gln                                                                              Leu                                                                              Ile                          ID. NO: 1)                                                                    PEB3 SEQ.                                                                            Asp                                                                              Val                                                                              Asn                                                                              Leu                                                                              Tyr                                                                              Gly                                                                              Pro                                                                              Gly                                                                              Gly                                                                              Pro                                                                              His                                                                              Thr                                                                              Ala                                                                              Leu                                                                              Lys                          ID. NO: 2)                                                                                       20             25             30                           PEB1   Val                                                                              Gly                                                                              Val                                                                              Lys                                                                              Asn                                                                              Asp                                                                              Val                                                                              Pro                                                                              His                                                                              Tyr                                                                              Ala                                                                              Leu                                                                              -- Asp                                                                              Gln                                                                              Ala                       PEB3   Asp                                                                              Ile                                                                              Ala                                                                              Ser                                                                              Lys                                                                              Tyr                                                                              Ser                                                                              Glu                                                                              Lys                                                                              Thr                                                                              Gly                                                                              Val                                                                              Lys                                                                              Val                                                                              Asn                                                                              Trp Asn Phe Gln           __________________________________________________________________________

Any sample suspected of containing C. jejuni or C. coli antibodies maybe tested in accordance with the methods set forth herein. Preferably,the samples to be tested are bodily fluids such as blood, serum, urine,tears, saliva and the like. Both medical and veterinary applications arecontemplated. In addition to human samples, samples may be taken frompoultry or mammals such as non-human primates, horses, swine, etc. Dueto the sensitivity of the test described, it is possible to dilute thesample prior to testing. Dilution may proceed by addition of any fluidcompatible with each of the sample, the antibodies to be tested, and theantigenic composition. Serum, when used as the sample, may, for example,be diluted with one or more fluids selected from the group consisting ofphosphate-buffered saline, pH 7.0-7.4 (hereinafter, "PBS")PBS-containing Tween 20 (hereinafter, "PBS T"), PBS T with thimerosal(hereinafter, "PBS TT") PBS TT (gelatin) (hereinafter, "PBS TTG"), andPBS TTG with bovine gamma globulin (hereinafter, "PBS TTGG") Dilutions,when testing for IgG antibody, may be as high as a ratio from about 1:00to about 1:1000, such as, for instance, about 1:800. When testing forIgA antibody, sample may be diluted, for example, about 1:25 to about1:200, such as 1:100, and for IgM antibody, about 1:50 to about 1:800(e.g. 1:200). IgG tests are preferred.

Preferred diluents and dilution ratios may vary according to the samplebeing tested. Urine, for instance, is already relatively dilute and maynot need to be diluted further. However, it may not be necessary toconcentrate urine as is often necessary with other assays. Prior totesting, the pH of urine is preferably adjusted to between about 7.0 and7.4, the preferred pH for antibody function.

While dilution of sample is not required, it is believed that dilutionreduces the possibility that significant antigen/antibody complexes willbe formed in the absence of C. jejuni- or C. coli-specific antibodies.The extent of dilution should be taken into account in adjusting thethreshold level of antigen/antibody complex which should be considered apositive signal.

While the present disclosure provides an easy method for obtaining thepreferred antigens from the deposited C. jejuni strain, it is emphasizedthat these antigens are common to a large number of C. jejuni strains asshown by their efficacy in testing for the existence of C. jejuni. Whilethe deposited strain and the description of the present specificationprovide an easy manner of isolating these antigens, it is emphasizedthat the present invention broadly encompasses use of these antigensregardless of the source from which they are derived.

Before contacting a test sample with antigenic compounds in accordancewith the invention it is preferred (but not necessary) that theantigenic composition be immobilized using conventional techniques. Inone alternative embodiment, liposome-based assays may be used asdescribed in more detail below. For conventional immoblization,polystyrene plates, for example, may be incubated with antigenicsuspensions made in accordance with the invention. Alternatively, forexample, antigens isolated as protein bands on electrophoretic gel maybe transferred to a nitrocellulose sheet by known methods (See Example3). See Towbin et al., Proc. Nat'l. Acad. Sci., 76: 4350-54 (1979);Burnette et al., Biochem., 112: 195-203 (1981). Numerous othertechniques are known in the art for binding antigens to substantiallyinert substrates.

Bound antigens in accordance with the invention are preferably contactedwith a dilute fluid which includes the sample to be tested for presenceof antibody to C. jejuni/C. coli. The antigen and sample are preferablyincubated for at least 5 to 15 minutes. Less time is needed whenincubation proceeds at or near human body temperature, about 37° C.Incubation at other temperatures, for instance 4° C., is also proper,but generally requires additional incubation time. Preferred incubationtime at 37° C. is from about 5 minutes to about 90 minutes. The boundantigens should then be rinsed to remove any unbound antibodies, i.e.,those which are not specific for the antigens. Preferably, rinsingproceeds with a buffer solution such as PBS T, PBS TT orTris/Tween/Sodium chloride/azide. Multiple rinsings are preferred.

During incubation, C. jejuni-specific antibodies bind to the immobilizedantigens to create antigen/antibody complexes. All unbound antibodiesare substantially removed during the rinsing procedure. Due to the highspecificity of the antigens of the invention, antibodies which are notspecific for C. jejuni/C. coli are substantially removed by the rinsing.Naturally, if the tested sample did not contain C. jejuni/C.coli-specific antibodies, the immobilized antigens would besubstantially free of human antibody, and subsequent testing forantigen/antibody complexes should not indicate a substantial presence ofsuch complexes. On the other hand, if the tested sample were rich in C.jejuni/C. coli-specific antibodies, these antibodies should have boundto the immobilized antigens to form a large quantity of antigen/antibodycomplex for subsequent detection.

Detection of antigen/antibody complex may be achieved by a wide varietyof known methods. Preferred methods include but are not limited toenzyme-linked immunosorbent assay, latex agglutination, Western-blottechnique or indirect immunofluorescence assay.

Typically, the C. jejuni/C. coli-specific antibodies complexed withimmobilized antigen are detected by contact with labelled or otherwisedetectable second antibodies specific for the immunoglobulin beingtested for. If the test sample is human sera, for example, thedetectable second antibody is specific for human immunoglobulin. Thelabelled second antibodies may be specific for any human antibody,preferably of the IgG or IgA type, most preferably IgG. When acutesero-conversion is suspected, an IgM test using a labelled secondantibody specific for IgM may be appropriate. The second antibodies arepreferably incubated with the immobilized antigens for about 5 minutesto about 2 hours, preferably 30 minutes to 60 minutes at a temperatureof about 20° C. to about 37° C. Then, the antigens are washed with abuffer solution (preferably multiple times) in order to remove allunbound labelled antibody. The washings will remove substantially alllabelled antibody except that which has bound to immunoglobulin presenton the antigens. Of course, substantially the only human immunoglobulinpresent at this point should be C. jejuni- or C. coli-specific antibody.Hence, the presence of C. jejuni- or C. coli-specific antibody may beindirectly measured by determining the presence or absence of thelabeled second antibody.

There are many known techniques for detecting the label, which vary withthe type of label used. For instance, fluorescein-labelled antibody maybe detected by scanning for emitted light at the characteristicwavelength for fluorescein. Alternatively, an enzyme label is detectedby incubation with appropriate substrate and detection of an enzymaticactivity, preferably activity resulting in a color change. Such activitycan be determined by visual inspection or can be read automatically by aspectrophotometer set at the appropriate wavelength.

For example, the enzyme label may be horseradish peroxidase and thesubstrate may be H₂ O₂ and2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) which produces inthe presence of the enzyme, a compound detectable by a spectrophotometerset at 414 nm.

In Western blotting, the positive signal may be detected when an enzymeis conjugated to the second antibody. Incubation with appropriatesubstrate enzymatically produces a color product in the immediatevicinity of the antigenic band resolved by this process. The presence ofa reactive band may be detected by visual inspection. See the proceduredescribed in Example 3. In an indirect immunofluorescence assay,fluorescein-labeled second antibodies may be detected byfluorescence-activated detectors, or by visual inspection.

A liposome-based assay may involve the presence of fluorescein, anenzyme or a substrate inside a liposome onto whose surface C. jejuniantigens are expressed. These liposomes are incubated with a dilutedbody fluid sample to be tested, and are thoroughly washed. Any liposomeswith immunoglobulins on their surface forming an antigen/antibodycomplex may be recognized by attaching a second antibody, specific tothe immuniglobulin being tested for, onto the inside walls of apolystyrene tube containing the liposomes. Liposomes having antibodybound to their surfaces will become immobilized on the tube walls, andnon-immobilized liposomes will be washed away. The liposomes can bylysed with, for instance, detergent, or complement, and the enzyme orsubtrate that was in the interior is now free to react with thecomplementary substrate (or enzyme) in the solution in the tube.Enzymatic activity, preferably a color change reaction could be detectedby visual inspection or spectrophotometric color determination.Enzymatic activity beyond the predetermined positive threshold indicatesthe presence of C. jejuni or C. coli specific antibodies.

Testing, of the invention with rabbit antiserum raised to strains notused in preparation of the antigenic mixture (heterologous) indicatedthat the antigenic composition could detect antibodies raised to thesestrains, as well as detecting antibodies raised to the homologousstrain. This indicated that the antigenic composition included conservedantigens and had the type of broad specificity which should be useful inserologic assays.

The sensitivity and specificity of the antibody detection in accordancewith the present invention have been determined using serum obtainedfrom persons from defined populations. Example 1 is illustrative of theresults of this assessment.

The invention is futher elucidated by reference to the followingexamples which are set forth only as non-limiting illustrations of theinvention.

EXAMPLE 1 Determination of the Antigenicity of the PEB1 and PEB3Proteins for Infected Humans Using an ELISA

The purified PEB1 and PEB3 proteins were compared with a crudeacid-extracted mixture of C. jejuni proteins as antigens recognized byhumans with diarrheal diseases. The ELISA system substantially followedthe teachings of Blaser et al. (Blaser et al., "Human serum antibodyresponse to Campylobacter jejuni as measured in an enzyme-linkedimmunosorbent assay," Infect. Immun., 1984;44:292-298) to detect humanserum IgG, except certain details were changed as described below.

To sensitize ELISA plates (Nunc, Inc., Naperville, Ill.), purifiedproteins and the crude surface protein preparation were diluted in0.015M carbonate buffer, pH 9.6. One hundred ul of these preparationswere added to each well, and the plates incubated at 4° C. overnight.The plates were washed once with 0.01M PBS, pH 7.2, in 0.03% Tween-20and 0.01% thimerosal (PBS-T-T) and were blocked with 200 ul/well of 0.1%gelatin in PBS-T-T overnight at 4° C. The plates were then washed twicewith PBS-T-T, and 100 ul of antisera diluted with 0.5% bovine gammaglobulin and 0.1% gelatin in PBS-T-T was added to each well. After a 1hour incubation at 37° C., the plates were washed four times as before,and 100 ul of peroxidase-goat-anti-human IgG (1:500, Boehringer MannheimBiochemical, San Diego, Calif.) or 100 ul of peroxidase goat-anti-rabbitIgG (1:5000) diluted with 0.1% bovine gamma globulin and 1% BSA inPBS-T-T was added to each well and incubated at 37° C. for 1 hour. Theplates were washed six times, 100 ul of peroxidase developer (20 mg of2,2'-azinobis[3-ethylbenzothiazoline-6-sulfonic acid], 34 ul of H₂ O₂,9.35 ml of 0.2M Na₂ HPO₄, and 10.65 ml of 0.1M citric acid) was added toeach well, and the product of the peroxidase reaction determined after15 minutes for human antibodies and 30 minutes for rabbit antibodies atroom temperature using a MR 600 microplate reader (Dynatech LaboratoriesInc., Alexandria, Va.) at 414 nm. 20 ng of antigen was used. Opticaldensities per well from acute and convalescent sera were compared.Seroconversion was defined as an Optical density 414 value inconvalescent serum greater than 50% that in acute serum. Seroconversionin 19 patients with sporadic cases of acute C. jejuni or C. colidiarrhea is shown below in Table 3:

                  TABLE 3                                                         ______________________________________                                        Seroconversion.sup.a to                                                       C. jejuni proteins of Campylobacter-infected                                  persons and persons with other diarrheal diseases                             Patient isolate                                                                            AE.sup.b                                                                              PEB1.sup.c                                                                            PEB2.sup.d                                                                          PEB3.sup.e                                                                          PEB4.sup.f                           ______________________________________                                        Campylobacter                                                                 jejuni/coli                                                                   1     C. coli    +       +     -     +     -                                  2     C. coli    +       +     -     +     +                                  3     C. jejuni  -.sup.g +     -     -.sup.g                                                                             +                                  4     C. coli    -.sup.g -.sup.g                                                                             -.sup.g                                                                             -.sup.g                                                                             -.sup.g                            5     C. jejuni  -       -     -     +     -                                  6     C. jejuni  +       +     -     +     +                                  7     C. jejuni  +       +     -     +     -                                  8     C. coli    +       +     -     +     -                                  9     C. jejuni  +       +     -     +     -                                  10    C. jejuni  +       +     -.sup. g                                                                            +     -                                  11    C. coli    +       +     -     +     -                                  12    C. jejuni  +       +     +     +     +                                  13    C. jejuni  +       +     -     +     -                                  14    C. jejuni  +       +     -     +     +                                  15    C. jejuni  -       -     -     +     -                                  16    C. jejuni  +       +     -     +     -                                  17    C. jejuni  +       +     +     +     +                                  18    C. jejuni  +       +     -     -     -                                  19    C. jejuni  -.sup.g -     -     -.sup.g                                                                             -                                  % seroconversion                                                                           73.7    78.9    10.5  78.9  31.6                                 Other pathogens                                                               1     Shigella   -       -     -     -     -                                  2     Salmonella -       -     -     -     -                                  3     Shigella   -       -     -     -     -                                  4     Yersinia   -       -     -     -     -                                  5     Salmonella -       -     -     -     -                                  % seroconversion                                                                           0       0       0     0     0                                    No pathogens                                                                  identified                                                                    1            -       -       -     -     -                                    2            -       -       -     -     -                                    3            -       -       -     -     -                                    4            -       -       -     -     -                                    5            -       -       -     -     -                                    % seroconversion                                                                           0       0       0     0     0                                    ______________________________________                                         .sup.a Patients had acute diarrheal illnesses for which they sought           attention at medical facilities in Denver. Acute phase sera were obtained     within seven days of illness onset and convalescent sera were obtained 11     to 40 days later. Seroconversion is defined as O.D.sub.414 value increase     by at least 50% in convalescent serum compared wth that in acute serun.       Value in Convalescent serun must be greater than 0.200. + =                   seroconversion, - = no seroconversion.                                        .sup.b Antigen was extracted from C. jejuni strain 81176 in glycine buffe     (pH 2.2) as described above (Acid extract).                                   .sup.c PEB1 antigen was purified to homogeneity fron acid extract through     hydrophobic interaction FPLC.                                                 .sup.d PEB2 antigen was purified to homogeneity from acid extract through     sequential cationic exchange, hydrophobic interaction, and gel filtration     FPLC.                                                                         .sup.e PEB3 antigen was purified to homogeneity from acid extract through     sequential cationic exchange and hydrophobic interaction FPLC.                .sup.f PEB4 antigen was purified to homogeneity from acid extract through     sequential cationic exchange and gel filtration FPLC.                         .sup.g O.D. value greater than 1.000 in both acute and convalescent serum                                                                              

EXAMPLE 2 Use of Specific Antiserum to the PEB1 C. jejuni ProteinAntigen in the Detection of Strains of C. jejuni and C. coli

We examined the potential application of an antisera to the C. jejuniPEB1 protein in the identification of C. jejuni and C. coli. Preparationof antisera and peformance of the ELISA followed previously describedmethods and as described in Example 1. Antisera against the mixture ofacid-extracted proteins, and the purified PEB1 protein, were raised inadult New Zealand white female rabbits by three subcutaneous injectionsat 2-week intervals of 5 ug of purified protein in 1 ml of an equivolumemixture of antigen and adjuvant (67% of 0.1M PBS at pH 7.2, 27%hexadecane, 6% glycerol). Fresh normal sera from the same rabbits wereobtained prior to inoculation. Thirty-five strains of C. jejuni, 15 ofC. coli, 10 of C. fetus, 5 of Campylobacter laridis and 5 ofHelicobacter pylori (formerly known as C. pylori) were used in thisstudy. The strains had been maintained frozen at -70° C. in brucellabroth containing 15% glycerol. For preparation of antigens, the cellswere grown overnight on blood agar plates, and harvested with cottonswabs and resuspended in water. Protein concentrations were determinedusing the Markwell et al. modification of the Lowry method;concentrations were adjusted to 1 ug/ml in water.

Whole bacterial cells (0.5 ug protein/well) were then used in an IgGELISA. The specific antisera were absorbed with whole Escherichia colibacterial cells, diluted 1:500 for antiserum to PEB1 and the mixture ofacid-extracted proteins, and tested in the ELISA system with an O.D.value greater than 0.1 at 414 nm. defined as positive. In this systemnormal rabbit serum did not recognize any of the Campylobacter strains,as expected (Table 4). In contrast, the antisera to the mixture ofacid-extracted proteins recognized all 35 C. jejuni strains, all 15 C.coli strains, 9 of 10 C. fetus strains, all 5 C. laridis strains, and 2of 5 H. pylori strains. The antiserum to the PEB1 protein recognized all35 C. jejuni strains, and all 15 C. coli strains, but none of the otherCampylobacter or Helicobacter isolates (Table 4 and FIG. 3). Thus, theantisera to the PEB1 protein appeared to have the greatestdiscriminatory power, having both 100% sensitivity and specificity forC. jejuni and C. coli.

                  TABLE 4                                                         ______________________________________                                        RECOGNITION OF CAMPYLOBACTER                                                  AND HELICOBACTER CELLS                                                        IN AN ELISA BY ANTISERA TO C. JEJUNI PROTEINS                                 % of Bacterial Strains Positive.sup.d                                                 C. jejuni                                                                              C. coli  C. fetus                                                                             C. laridis                                                                           H. pylori                             Antisera                                                                              (n = 35) (n = 15) (n = 10)                                                                             (n = 5)                                                                              (n = 5)                               ______________________________________                                        NRS.sup.a                                                                              0        0        0      0      0                                    anti-AE.sup.b                                                                         100      100      90     100    40                                    anti-PEB1.sup.c                                                                       100      100       0      0      0                                    ______________________________________                                         .sup.a Normal rabbit serum absorbed with E. coli bacterial cells              .sup.b Antisera from adult NZW rabbits hyperimmunized with pooled             acidextracted C. jejuni antigens as described in the methods section          above, subsequently absorbed with E. coli bacterial cells                     .sup.c Antisera from adult NZW rabbits hyperimmunized with the purified       PEB1 antigen (prepared as described above) as described in the methods        sections above, subsequently absorbed with E. coli bacterial cells            .sup.d All bacterial strains used were clinical isolates of Campylobacter     and Helicobacter species that had been stored at -70° C. prior to      subculture and testing.                                                  

EXAMPLE 3 Detection of PEB1 Antigen in Whole Bacterial Cells by WesternBlot

To further confirm the specificity of recognition of Campylobacterstrains in ELISA by antiserum to PEB1 antigen in Example 2, we performedWestern blotting to examine for the bands recognized by this serum inpreparations of whole bacterial cells of various Campylobacter andHelicobacter species. Whole bacterial cells were prepared as describedin Example 2. 0.5 mg of bacterial protein was loaded in each lane inSGS-PAGE with 15% acrylamide. The method of Towbin et al. [Proc. Nat'l.Acad. Sci., 76:4350-54 (1979)] formed the basis of the Western blotprocedure we used. In brief, SDS-PAGE was performed with a gel thicknessof 0.75 mm in a mini-protean II dual slab cell (Bio-Rad Laboratories,Richmond, Calif.) at 250 mA for about 40 minutes. The proteins were thentransferred from the slab gel to nitrocellulose paper by electroblottingfor 30 minutes at 1000 mA. The nitrocellulose paper was then blockedonce for 30 minutes in Tris/saline blotting buffer (TSBB) (10 mM Trisbase, pH 8.0, 0.5M NaCl, 0.5% Tween 20, 0.02% NaN₃). The nitrocellulosepaper was incubated with a 1:2000 dilution of antiserum to PEB1 fromstrain 81-176 in TSBB for 60 minutes. After three washes in TSBB, thenitrocellulose paper was incubated with 1:2000 dilution of Alkalinephosphatase-conjugated anti-rabbit IgG (Amersham Corp., ArlingtonHeights, Ill.) for 60 minutes. After washing, the nitrocellulose paperwas developed in substrate solution containing 9 ml of 3 mM MgCl₂ in 50mM tris, pH 10.0, 1 ml of 0.1% nitrobluetetrazolium and 0.1 m. of 0.5%of 5-bromo-4-chloro-3-indoxlyphosphate (Sigma, St. Louis, Mo.) indimethyl formamide. In total, 18 C. jejuni strains, 14 C. coli, 3 C.fetus, 4 C. laridis strains and 1 H. pylori strain were tested. A 28 kDaband was found in all 18 C. jejuni and all 14 C. coli strains, but notfound in any of the C. fetus, C. laridis, or H. pylori strains tested(FIG. 3). Thus, this Western blot experiment provided physical evidencethat PEB1 antigen from various C. jejuni/coli strains are allantigenically related and can be recognized by antiserum to PEB1 antigenfrom a single strain, 81-176 (ATCC55026). This specificity forms thebasis for using PEB1 protein and antibody to this protein in diagnosisof C. jejuni/coli infection. In addition to confirming the reliabilityof ELISA results in EXAMPLE 2, the Western blot shown here is anotheruseful tool in diagnosis.

EXAMPLE 4 Digestion of PEB1 Antigen with Proteinase K

An important question to resolve is whether PEB1 antigen is a protein.To answer this, 24-hour cultures of Campylobacter strains on blood agarplates were harvested in sterile distilled H₂ O (5 ml/plate). The cellswere pelleted at 3500 g for 10 minutes and resuspended in H₂ O. Theprotein concentration was determined using the BCA protein assay kit(Pierce, Rockford, Ill.) and adjusted with H₂ O to 240 ug/ml. For enzymedigestion, 2.4 ug of proteinase K (1 ug/ul) was added to 100 ul ofbacterial suspension. Proteinase K was replaced by H₂ O in controldigestions. The mixture was incubated at 37° C. in a water bath for 60minutes, then 100 ul of SDS-sample buffer was added. The samples weremixed and boiled for 5 minutes, then kept at room temperature to cool,and then run on SDS-PAGE with 15% acrylamide. 8 ul of sample was loadedper lane. Western blot was performed as described in EXAMPLE 3. Afterelectrophoresis, the gel was transferred onto nitrocellulose paper. Thenitrocellulose paper was blocked and then reacted with rabbit-anti PEB1.The second antibody was alkaline phosphatase conjugated-goat-anti-rabbitIgG. After washing, the nitrocellulose paper was developed to visualize28 kDa bands as described in EXAMPLE 3. The 28 kDa band disappearedafter proteinase K digestion, but not after the control digestion of allfour C. jejuni/coli strains and of the glycine extract of C. jejunistrain 81-176. This indicates that the major antigenic component of PEB1antigen is a protein (FIG. 2).

EXAMPLE 5 Determination of Native Molecular Weight of PEB1 Antigen

The native molecular weight of PEB1 antigen was determined in a Superose12 (Pharmacia LKB, Piscataway, N.J.) gel filtration column using gelfiltration molecular weight markers (Sigma, St. Louis, Mo.), assigned asfollows: horse spleen apoferritin (443,000), sweet potato beta-amylase(200,000), yeast alcohol dehydrogenase (150,000), bovine erythrocytecarbonic anhydrase (29,000) and horse heart cytochrome C(12,400). Bluedextran (2,000,000) was used to determine the void volume. Individualprotein standards were dissolved in a equilibration buffer containing 50mM tris-HCl, 100 mM KCl, pH7.5. Glycine extracts of C. jejuni/coli weredialyzed against water to remove glycine using the Centricon-10 (Amicon,Danvers, Mass.) microconcentrator. First water extracts of C.jejuni/coli strains were concentrated in the same way as for the glycineextracts. These samples were diluted 1:1 with the tris-KCl buffer, theneither 50 ul of sample or a molecular weight protein standard was loadedinto the column. Elution volume of the standards was individuallydetermined by the position of the absorption peak at 280 nm. A standardcurve of molecular weight was generated by semilog regression of elutionvolume versus Log₁₀ molecular weight of the protein standard. Elutionvolume of PEB1 antigen was determined by checking for the presence ofthe 28 kDa band in each fraction using SDS-PAGE and Western blot withrabbit anti-PEB1 as described in EXAMPLE 3. Two C. jejuni (strains81-176, D1916) and two C. coli (strains D743 and D1035) strains wereused in this study. In glycine extracts, PEB1 antigen was consistentlyeluted off the column immediately after the molecular weight standardprotein carbonic anhydrase (29 kDa) (FIG. 3), and had a calculatedmolecular weight of 28.9 kDa, indicating that the PEB1 antigen is amonomer. To compare effect of conditions for extraction onpolymerization of PEB1 antigen, we included water extracts in thisstudy. Only trace amounts of PEB1 antigen was extracted in water, so thewater extract was concentrated 50-fold by Centriprep-10 (Amicon)concentrator before use. PEB1 antigen extracted in water also was foundto be a monomer of 28.9±1.0 kDa for each of four strains tested. Inconclusion, the native form of PEB1 antigen was a monomer of 28.9±1.0kDa, a comparable value to that determined by SDS-PAGE under reducingand denaturing condition (28 kDa). The PEB1 antigens in C. jejunistrains have the same native molecular weights as those in C. colistrains.

EXAMPLE 6 Preparation of an Oral Vaccine for Administration to Mammalsor Poultry

We have considered the potential application of the use of the PEB1and/or PEB3 antigens in the development of a vaccine against C. jejuniand C. coli infections. To limit the effects of gastric acid andproteolytic enzymes on the vaccine preparation, the whole PEB1 and/orPEB3 antigen (or a fragment of one or both of these proteins) will bepackaged either in an enteric coated gelatin capsule or administeredwith sodium bicarbonate (Black et all, "Immunogenicity of Ty21aattenuated Salmonella typhi given with sodium bicarbonate or inenteric-coated capsules." Dev. Biol. Stand. 53:0, 1983). Dosage foradult humans preferrably varies from 5.0-50.0 mg of the antigens of theinvention, which may be either pure PEB1, pure PEB3, or a mixture ofPEB1 and PEB3, for example, a dosage of about 10.0 mg of pure or mixedantigen.

To enhance delivery of PEB1 and/or PEB3 to the gastrointestinal immunesystem the protein(s) [or a fragment(s) of the proteins] may beincorporated without chemical coupling into biodegradable microspheresthat are 5-10 μm in size that will be ingested orally (Eldridge et al.,"Biodegradable microsphere: vaccine delivery systems for oralimmunization," Curr. Top. Microbiol. Immunol. 146:59, 1989). Themicrospheres are composed of co-polymers of glycolic and lactic acidswhich are degraded into original components by hydrolysis. Adjusting theratio of glycolic to lactic acids within the co-polymers varies the rateof hydrolysis from several hours to several months. Thus, both fast- andslow-releasing microspheres can be created. The use of a mixture of bothfast- and slow-releasing microspheres will then be used to allow forinduction of both a primary and secondary immune response with a singleoral immunization.

EXAMPLE 7 Preparation of a Parenteral Vaccine for Administration toMammals or Poultry

Although for gastrointestinal pathogens, orally administered vaccinesappear to be preferable, for several other infectious agents, parenteralvaccine show efficacy. A component of the bacterium Salmonella typhi,the cause of typhoid fever, has been purified and used as aparenteral-administered vaccine. This component, the Vi capsularpolysaccharide, is highly efficacious (Klugman K. P., et al. Protectiveactivity of Vi capsular polysaccharide vaccine against typhoid fever,"Lancet 1987;2:1165-69"). The Salk vaccine for polio is administeredparenterally and it prevents the disease of polio, although havinglittle or no effect on becoming infected with the polioviruses.Parenteral vaccines also have efficacy, although limited, in preventingcholera.

For C. jejuni, a parenteral vaccine could include PEB1 and/or PEB3 orfragments thereof. The protein(s) or fragment(s) could be administeredwith an adjuvant or by itself in a suitable buffer. Reasonable adjuvantsinclude, but are not limited to, muramyl dipeptide, concanavalin A, DEAEdextran, lipid polyvalent cations, or hydrocarbons such as hexadecane.

C. jejuni vaccine could be given to humans as 1.0 mg (range 0.5-5.0 mg)of antigen (PEB1, PEB3, or mixture of both) in 1 ml of phosphatebuffered saline (pH7.4). With a suitable antigen, only a single dose maybe needed, but multiple doses with or without adjuvants could beconsidered.

EXAMPLE 8

C. jejuni/C. coli-specific test kits are constructed for detectingantibodies using several different techniques for detection. One testkit for antibody detection comprised of a compartmented enclosurecontaining a plurality of wells, plates which were coated prior to usewith PEB1 or PEB3 antigens, and ELISA materials for enzyme detectionconsisting of peroxidase-labeled goat anti-human IgG and a color changeindicator consisting of ABTS in McIlvain's buffer with 0.005 percenthydrogen peroxide. Naturally, other enzymes and developers could havebeen used. For instance, alkaline phosphatase-labelled goat anti-humanIgG could be used in conjunction with p-nitrophenyl phosphate indiethanolamine and magnesium chloride buffer.

A second test kit for detecting antibodies using the Western blottechnique is comprised of a container, cover, nitrocellulose sheet, anda polyacrylamide slab gel in the presence of sodium dodecyl sulfate,surfactants, pH modifiers, dried non-fat milk and materials for enzymedetection including a color change indicator consisting of DAB in Triswith hydrogen peroxide. This Western blot analysis kit also containsperoxidase-labelled goat or rabbit anti-human immunoglobulin and asource of PEB1 or PEB3 antigens.

Another C. jejuni/C. coli-specific test kit for detecting antibodiesusing the indirect immunofluorescence assay may include a compartmentalcontainer with PEB1 or PEB3 antigens, human test serum, phosphatebuffered saline and fluorescein-conjugated goat anti-human IgG.

Finally, a different C. jejuni/C. coli specific test kit for detectingantibodies uses liposomes and comprises a container, human test serum,fluorescent marker- (or enzyme- or substrate-) filled liposome with C.pylori antigens on their surface, and a surface-active agent. In thisassay the container mght be a precoated tube or well with goatanti-human IgG.

C. jejuni/C. coli-specific test kits are constructed for detecting C.jejuni or C. coli cells using several different techniques fordetection. One test kit for detection of C. jejuni or C. coli cellscomprises a compartmented enclosure containing a plurality of wells,plates that could be coated with cells of the bacterial strain to betested, a hyperimmune antiserum to PEB1 antigen, and appropriate ELISAmaterials such as those discussed above in this example.

A second test kit for detecting C. jejuni or C. coli cells using theWestern blot technique is comprised of a container, cover,nitrocellulose sheet, and a polyacrylamide slab gel in the presence ofsodium dodecyl sulfate, surfactants, pH modifiers, dried non-fat milkand materials for enzyme detection including a color change indicatorconsisting of DAB in Tris with hydrogen peroxide. This Western blotanalysis kit also contains goat anti-rabbit immunoglobulin and a sourceof hyperimmune antiserum to PEB1.

Another C. jejuni/C. coli-specific test kit for detecting antibodiesusing the latex agglutination assay may include a compartmentalcontainer, hyperimmune serum to PEB1 and/or PEB3 conjugated to latexbeads, and phosphate buffered saline or water.

The kits described above could be utilized for detection of C. jejuni orC. coli organisms in fecal or water specimens, fecal or water specimensenriched for Campylobacter by selective enrichment methods, or incolonies on solid media suspected as being C. jejuni or C. coli.

The terms and descriptions used herein are preferred embodiments setforth by way of illustration only, and are not intended as limitationson the many variations which those of skill in the art will recognize tobe possible in practicing the present invention as defined by the patentclaims based on the present disclosure.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 2                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                         (v) FRAGMENT TYPE: N-terminal                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GlyGluGlyLysLeuGluSerIleLysSerLysGlyGlnLeuIleVal                              151015                                                                        GlyValLysAsnAspValP roHisTyrAlaLeuXaaAspGlnAla                                202530                                                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       AspValAsnLeuTyrGlyProGlyGlyProHisThrAlaLeuLysAsp                              151015                                                                         IleAlaSerLysTyrSerGluLysThrGlyValLysValAsnTrpAsn                             202530                                                                        PheGln                                                                    

What is claimed is:
 1. Antisera raised against a purified PEB1 antigenwhich is obtained from Campylobacter jejuni and has an apparentmolecular weight of 28 kDa as measured on sodium dodecyl sulfatepolyacrylamide gel under reducing conditions, a molecular weight of28.9±1.0 kDa as measured by gel filtration chromatography under nativeconditions, and an isoelectric point of 8.5.